Project Summary Zika virus (ZIKV) is a mosquito-borne virus that causes severe neurological diseases such as microcephaly in neonates and Guillain-Barre syndrome in adults. ZIKV is also a global threat with a large at-risk population in the tropical and subtropical regions. Furthermore, non-vector borne transmission allows ZIKV to infect people worldwide. There are neither direct-acting antivirals nor vaccines available against Zika. The ZIKV NS2B-NS3 protease is an attractive therapeutic target because of its essential role in viral maturation. The current peptidomimetic inhibitors of the ZIKV protease suffer from poor cellular potency and nonselective inhibition of human proteases. The limitations of these peptidomimetic inhibitors are a result of their peptide scaffold and covalent warhead groups used to increase their potency. Hence, there is a need to discover clinically viable protease inhibitors (PIs) to generate lead compounds for further drug development. Fragment-based drug design (FBDD) is a promising approach for discovering novel scaffolds to overcome the limitations of peptidomimetics. This project aims to identify clinically viable ZIKV PIs using FBDD. I hypothesize that FBDD will result in potent and selective ZIKV PIs. Moreover, how the ZIKV protease recognizes diverse substrates remains unknown. Similar observations of diverse substrates recognition by other viral proteases led to the discovery of the substrate envelope (SE) model, which states that the conscience volume the diverse substrates occupy within the active site of a protease is the mode of recognition. I hypothesize that the SE model can explain diverse substrate recognition by the ZIKV protease. The SE model also explained the structural basis of drug resistance for those viral proteases. PIs that bind within the consensus volume of the substrates, i.e. the substrate envelope, are less susceptible to drug resistance. Drug resistance is the biggest problem facing direct-acting antivirals targeting viral proteases of other highly mutating viruses. Therefore, the problem of drug resistance must be addressed at the early stages of drug discovery. This proposal allows the unique opportunity to incorporate the ZIKV SE model into the FBDD approach to preemptively address the problem of drug resistance. Hence, the ZIKV SE can be a structural tool to guide inhibitor design. To investigate these hypotheses, I will leverage my expertise in medicinal chemistry and structural biology to rationally design, synthesize and evaluate the ZIKV PIs. I will rely on my experience in crystallography and computational biology to solve co-crystal structures of the ZIKV protease with inhibitors and substrates, respectively. From the protease-substrate complex structures, I will generate a ZIKV protease SE. The results from this project will provide an understanding of the structural basis of diverse substrate recognition by the ZIKV protease, and also generate clinically viable PIs for further drug development.